25
4 DRINKING WATER DISTRIBUTION SYSTEMS: BIOFILM MICROBIOLOGY 4.1 INTRODUCTION Following water treatment by processes mentioned in Chapters 2 and 3, water is dis- tributed through a network of pipes to reach customers. The water distribution system (WDS), an essential component of drinking water treatment, is the “workhorse” that carries drinking water from the plant to the customers. The daily production in the United States is estimated 34 billion gallons of drinking water that flows through 1.8 million miles of distribution pipes. It is estimated that approximately two-third of the pipes are made of plastic, mostly polyvinyl chloride (PVC). Many waterborne outbreaks are attributed to the degradation of water quality in noncovered water reser- voirs and in WDSs through cross-connections, main breaks (an estimated 240,000 water distribution main breaks per year in the United States; Lafrance, 2011), con- tamination during construction or repair, back siphonage (National Research Council, 2006), or negative pressure events (Gullick et al., 2004; NRC, 2006). Drinking water regulations mostly concerns water quality at the end of the water treatment plant but the water quality can deteriorate during storage and transport through water distribution pipes and in indoor plumbing before reaching the con- sumer. More recently, US federal regulations addressed water quality in distribution systems through rules covered by the Safe Drinking Water Act (SDWA). In the long history of water distribution which goes back to 2000 BC, pipes were made of a wide range of materials which include aqueducts, terracotta, wood, copper, lead, bronze, bamboo, and stone. Nowadays, piping materials include cast iron, ductile iron, stainless steel, concrete, asbestos-cement, or PVC, polyethylene, and medium density polyethylene (MDPE) (Bachmann and Edyvean, 2006; Berry et al., 2006; Lafrance, 2011; Walski, 2006; Percival et al., 2000). However, some of Microbiology of Drinking Water Production and Distribution, First Edition. Gabriel Bitton. © 2014 John Wiley & Sons, Inc. Published 2014 by John Wiley & Sons, Inc. 91

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Page 1: Microbiology of Drinking Water (Production and Distribution) || Drinking Water Distribution Systems: Biofilm Microbiology

4DRINKING WATERDISTRIBUTION SYSTEMS:BIOFILM MICROBIOLOGY

4.1 INTRODUCTION

Following water treatment by processes mentioned in Chapters 2 and 3, water is dis-

tributed through a network of pipes to reach customers. The water distribution system

(WDS), an essential component of drinking water treatment, is the “workhorse” that

carries drinking water from the plant to the customers. The daily production in the

United States is estimated 34 billion gallons of drinking water that flows through 1.8

million miles of distribution pipes. It is estimated that approximately two-third of

the pipes are made of plastic, mostly polyvinyl chloride (PVC). Many waterborne

outbreaks are attributed to the degradation of water quality in noncovered water reser-

voirs and in WDSs through cross-connections, main breaks (an estimated 240,000

water distribution main breaks per year in the United States; Lafrance, 2011), con-

tamination during construction or repair, back siphonage (National Research Council,

2006), or negative pressure events (Gullick et al., 2004; NRC, 2006).

Drinking water regulations mostly concerns water quality at the end of the water

treatment plant but the water quality can deteriorate during storage and transport

through water distribution pipes and in indoor plumbing before reaching the con-

sumer. More recently, US federal regulations addressed water quality in distribution

systems through rules covered by the Safe Drinking Water Act (SDWA).

In the long history of water distribution which goes back to 2000 BC, pipes

were made of a wide range of materials which include aqueducts, terracotta, wood,

copper, lead, bronze, bamboo, and stone. Nowadays, piping materials include cast

iron, ductile iron, stainless steel, concrete, asbestos-cement, or PVC, polyethylene,

and medium density polyethylene (MDPE) (Bachmann and Edyvean, 2006; Berry

et al., 2006; Lafrance, 2011; Walski, 2006; Percival et al., 2000). However, some of

Microbiology of Drinking Water Production and Distribution, First Edition. Gabriel Bitton.© 2014 John Wiley & Sons, Inc. Published 2014 by John Wiley & Sons, Inc.

91

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92 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

the piping materials lead to corrosion problems (e.g., cast iron and copper pipes) or

to leaching of toxic materials (e.g., PVC pipes).

Some major problems concerning WDSs are the following:

� Microbial growth� Pathogen survival and growth� Nitrification problems� Biocorrosion

Deterioration of water distributions systems due to leakage from cracks and joints

or back siphonage may lead to drinking water contamination and disease outbreaks.

In the United States, approximately 18% of the outbreaks reported for public water

systems are due to contamination of the WDS by microbial pathogens and toxic

chemicals (Craun, 2001). The contribution of WDSs to acute gastrointestinal illness

(AGI) incidence was demonstrated by epidemiological studies which showed that the

incidence was higher among consumers who drank from home taps than those who

drank treated water bottled at the water treatment plant (Payment et al., 1991a, 1997).

This shows that WDS and in-premise plumbing may contribute to disease in the

community. The risk of viral AGI from nondisinfected groundwater-based drinking

WDSs exceeded the U.S. EPA acceptable risk of 10−4 episodes/person-year. An

extrapolation of the results to the United States as a whole shows an AGI risk of

470,000 to 1,100,000 episodes/year nationwide (Lambertini et al., 2012; U.S. EPA,

1989).

Drinking water deterioration inWDS has several causes (LeChevallier et al., 2011;

Smith, 2002; Sobsey and Olson, 1983):

� Improperly built and operated storage reservoirs which should be covered to

prevent airborne contamination and to exclude animals.� Loss of disinfectant residual.� Back siphonage and cross-contamination.

Some of the consequences are

� Public health problems due to the excessive growth and colonization ofwater dis-

tribution pipes by bacteria and other organisms, some of which are pathogenic.

Microbial growth depends on the availability of nutrients (see Chapter 6)� Microbial regrowth in storage reservoirs� Protection of pathogens from disinfectant action� Taste and odor problems due to the growth of algae, actinomycetes and fungi

(see Chapter 5)� Corrosion problems

Drinkingwatermay contain humic and fulvic acids, aswell as easily biodegradable

natural organics such as carbohydrates, proteins, and lipids. The presence of dissolved

organic compounds in finished drinking water is responsible for several problems,

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BIOFILM DEVELOPMENT IN WDSs 93

among which are taste and odors, enhanced chlorine demand, trihalomethane forma-

tion, and bacterial colonization of water distribution lines (see Chapter 6 for more

details). Water utilities should maintain the distribution system by periodically flush-

ing the lines to remove sediments, excessive bacterial growth, and encrustations due

to corrosion.

4.2 BIOFILM DEVELOPMENT IN WDSs

4.2.1 Introduction

Biofilms develop at solid-water interfaces and are widespread in natural environments

as well as in engineered systems. They are ubiquitous and are commonly found in

trickling filters, rotating biological contactors, activated carbon beds, distribution pipe

surfaces, groundwater aquifers, aquatic weeds, tooth surfaces (i.e., dental plaque),

dialysis units, dental unit waterlines, and indwelling medical devices (IMDs) such

as catheters, pacemakers, orthopedic implants, endotracheal tubes, prosthetic joints,

and heart valves (Anwar and Costerton, 1992; Characklis, 1988; Hamilton, 1987;

Schachter, 2003; Thomas et al., 2004; Walker and Marsh, 2007; van der Wende

and Characklis, 1990). For example, in the United States, the risk of infection from

bladder catheters ranges from 10% to 30% while the risk from cardiac pacemakers

is 1–5% (Thomas et al., 2004).

Biofilms are relatively thin layers (up to a few hundred microns thick) of attached

microorganisms that form microbial aggregates and grow on surfaces. Biofilms

formed by prokaryotic microorganisms resemble in some ways tissues formed by

eukaryotic cells (Costerton et al., 1995; Wingender and Flemming, 2011). The multi-

cellular behavior is due to the fact that cells within the biofilm interact and coordinate

their activities via direct cell-to-cell contact or by releasing signaling molecules

(see quorum sensing in Section 4.2.5) (Branda and Kolter, 2004). Observations of

living biofilms by scanning confocal laser microscopy have helped in our understand-

ing of biofilm structure. Biofilm microorganisms form microcolonies separated by

open water channels (Stoodley et al., 2002; Donlan, 2002). Liquid flowing through

these channels allow the diffusion of nutrients, oxygen, and antimicrobial agents

into the cells (Donlan, 2002). Some bacteria, such as Staphylococcus epidermidis,form honeycomb-like structures (Schaudinn et al., 2007) that provide mechanical

stability to the biofilm, help lower the energy costs of individual cells and maximize

nutrient absorption. Biofilms also include corrosion by-products, organic detritus,

and inorganic particles such as silt and clay minerals. They contain heterogeneous

assemblages of microorganisms (Figure 4.1), depending on the chemical composition

of the surface, the chemistry of finished water, and oxido-reduction potential in the

biofilm. They take days to weeks to develop, depending on nutrient availability and

environmental conditions. Biofilm growth proceeds up to a critical thickness (approx-

imately 100–200 μm) when nutrient diffusion across the biofilm becomes limiting.

The decreased diffusion of oxygen is conducive to the development of facultative and

anaerobic microorganisms in the deeper layers of the biofilm.

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94 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

Figure 4.1 SEM pictures of biofilms.

4.2.2 Processes Involved in Biofilm Development

A number of processes contribute to biofilm development on surfaces exposed to

water flow. The processes involved are the following (Bitton and Marshall, 1980;

Gomez-Suarez et al., 2002; Olson et al., 1991). The sequence of events is described

in Figures 4.2 and 4.3 (Gomez-Suarez et al., 2002; De Kievit, 2009):

4.2.2.1 Surface Conditioning. Surface conditioning is the first step in biofilm

formation. Minutes to hours after exposure of a surface to water flow, a surface-

conditioning layer, made of ions, proteins, glycoproteins, humic-like substances, and

other dissolved or colloidal organic matter, initially adsorb to the surface. This results

in a modified surface that is different from the original one (Schneider and Leis,

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BIOFILM DEVELOPMENT IN WDSs 95

(a) (b)

(c) (d)

Conditioning film Transport

Initial adhesion Co-adhesion

(e) (f)Anchoring

Yeasts Bacteria Polymers Conditioning film Surface structures

Growth

Figure 4.2 Sequence of events in biofilm formation Source: Gomez-Suarez et al. (2002).In: Encyclopedia of Environmental Microbiology, Gabriel Bitton, editor-in-chief, Wiley-

Interscience, N.Y.

2002). The conditioning film is a source of nutrients for bacteria, particularly in

oligotrophic environments such as drinking water or groundwater.

4.2.2.2 Transport of Microorganisms to Conditioned Surfaces. Diffu-

sion, Brownian motion, convection, and turbulent eddy transport are involved in

turbulent flow regime. Chemotaxis may also enhance the rate of bacterial adsorption

to surfaces under more quiescent flow.

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96 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

1

2 3 4

5

Figure 4.3 Pseudomonas aeruginosa biofilm development. Planktonic cells (stage 1) attach

onto a solid surface (stage 2) and microcolonies are formed (stage 3). Under conditions that

promote bacterial migration (e.g., succinate, glutamate), cells will spread over the substratum,

ultimately developing into a flat, uniformmat (stage 4). Undermotility-limiting conditions (e.g.

glucose), the microcolonies proliferate forming stalk- and mushroom-like structures (stage 4).

At various points throughout biofilm maturation, cells can detach and resume the planktonic

mode of growth (stage 5). Source: De Kievit, T.R. (2009). Environ. Microbiol. 11: 279–288.

4.2.2.3 Adhesion of Microorganisms to Surfaces. According to the ther-

modynamic theory, adhesion of a microorganism to a surface is favored when the

free energy of adhesion is negative (ΔGadh < 0) (Gomez-Suarez et al., 2002). Fur-

thermore, according to the DLVO theory (Derjaguin, Landau, Verwey, and Overbeek

theory), adhesion is a balance between Lifshitz-Van der Waals forces and repulsive

or attractive electrostatic forces due to electrical surface charges on both microbial

and surfaces. Hydrophobic interactions are also involved in microbial adhesion to

surfaces.

4.2.2.4 Cell Anchoring to Surfaces. Following adhesion to a given surface,

microbial cells are anchored to the surface, using extracellular polymeric substances

(EPSs). EPS, made of polysaccharides (e.g., mannans, glucans, uronic acids), pro-

teins, nucleic acids, and lipids help support biofilm structure. EPS display hydrophilic

and hydrophobic properties. They help in the adhesion of microorganisms to surfaces

and their cohesion within biofilms, and play a structural role in biofilms (Bitton

and Marshall, 1980; Characklis and Cooksey, 1983; Costerton and Geesey, 1979;

Flemming and Wingender, 2002; 2010; Starkey et al., 2004; Zhu et al., 2010; Xue

et al., 2013b). EPS also helps protect microorganisms from protozoan predation,

chemical insult, antimicrobial agents, desiccation, and osmotic shock. Moreover,

extracellular polysaccharides chelate heavy metals and reduce their toxicity to

microorganisms (Bitton and Freihoffer, 1978).

Other attachment organelles include flagella, pili (fimbriae), stalks, or holdfasts

(e.g., Caulobacter) (Bitton and Marshall, 1980; Olson et al., 1991).

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BIOFILM DEVELOPMENT IN WDSs 97

4.2.2.5 Cell Growth and Biofilm Accumulation. Biofilms essentially con-

sist of microbial aggregates embedded in EPSs attached to a solid surface (Rittmann,

1995b). It was estimated that biofilms harbor about 95% of the microbial communi-

ties in WDSs (Wingender and Flemming, 2004). However, bulk water bacteria may

dominate in portions of the distribution system with no chlorine residual (Srinivasan

et al., 2008).

4.2.3 Factors Involved in Biofilm Accumulation

The study of single-species biofilms has helped in our understanding of the steps

involved in biofilm accumulation which depends on several factors:

4.2.3.1 Concentration of Assimilable Organic Carbon. The presence of

even microgram levels of organic matter in distribution lines allows the growth and

accumulation of biofilm microorganisms (Block et al., 1993; Ollos et al., 2003;

Servais, 1996).

4.2.3.2 Limiting Nutrients. Microbial growth in drinking water can also be

highly regulated by the availability of phosphorus, which can act as a limiting nutrient

for microbial growth in drinking water (Charnock, andKjønnø, 2000;Miettinen et al.,

1997; Keinanen et al., 2002; Sathasivan and Ohgaki, 1999). For example, in most

Finnish drinking waters, microbial growth was well correlated with the concentration

of bioavailable phosphorus (Lehtola et al., 2002; Rubulis and Juhna, 2007). The

presence of bioavailable phosphorus and iron in water distribution pipes also helps

the survival of Escherichia coli (Appenzeller et al., 2005; Juhna et al., 2007). Thus,phosphorous removal by water treatment processes may decrease E. coli survival inthis environment.

4.2.3.3 Trace Elements. Trace elements can stimulate (e.g., Fe, Mn, Zn) or

inhibit (e.g., Cu, Ag) bacterial growth in distribution networks.

4.2.3.4 Disinfectant Concentration. Biofilm thickness and activity are inhib-

ited by disinfection with chlorine. For example, the biofilm thickness (∼103 pg

ATP/cm2) in the presence of a chlorine concentration of <0.05 mg/L was two orders

of magnitude greater than the thickness (∼10 pg ATP/cm2) in the presence of a chlo-

rine level of 0.30 mg/L (Hallam et al., 2001). Attached microorganisms are better

controlled by monochloramine than by free chlorine (see also Chapter 3). In a bench-

scale distribution system, it was found that a free chlorine residual of 0.5 mg/L or

chloramine at 2mg/L reduced biofilmmicroorganisms by several orders ofmagnitude

(Ollos et al., 2003).

4.2.3.5 Type of Pipe Material. Biofilm thickness also depends on the type of

pipe material. Plastic-based materials (polyethylene or PVC) support less attached

biomass than iron (e.g., gray iron), cement-based materials (e.g., asbestos-cement,

cemented cast iron) (Figure 4.4; Niquette et al., 2000), or stainless steel materials.

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98 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

Figure 4.4 Effect of pipe material on biofilm accumulation in a drinking water distribution

system. Adapted from Niquette et al. (2000). Water Res. 34: 1952–1956.

Copper pipes showed the lowest biofilm formation potential (Yu et al., 2010). Biofilm

formation, as measured by heterotrophic plate counts and ATP content, was higher

in galvanized steel pipes than in copper pipes, as the release of Cu from the pipes

is toxic to microorganisms (Silhan et al., 2006). Iron corrosion, increased surface

roughness, and porosity in iron pipes contribute to increased attached biomass. Iron,

as a nutrient, is also a factor in increased biomass in the pipes. Pipe material type can

also influence the community composition in the biofilms (Kalmbach et al., 2000).

For example, 74% of the bacteria in PVC pipes were Stenotrophomonas, whereasiron pipes harbored a more diverse microbial populations which consisted mainly of

Nocardia, Acidovorax, Xanthobacter, Pseudomonas, and Stenotrophomonas (Nortonand LeChevallier, 2000). Thus, the use of pipe materials (e.g., stainless steel) that

could reduce biofilm development is desirable (Percival et al., 2000).

4.2.3.6 Flow Velocity and Regimen. An increase in flow rate and a change

from laminar to turbulent flow can enhance the mass transport of nutrients or biocides

into the biofilm, leading to changes in biofilm thickness (see Bachmann and Edyvean

(2006) for more details). Sudden changes in water flow leads to detachment of biofilm

from the pipe surface with subsequent deterioration of water quality due to increase

in suspended bacteria (Choi and Morgenroth, 2003). Hydraulic regime also affects

the bacterial community structure in biofilms. A higher diversity was observed at

highly varied flow in an experimental WDS (Douterelo et al., 2013).

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BIOFILM DEVELOPMENT IN WDSs 99

4.2.3.7 Quorum Sensing. A bacterial communication system, which controls

biofilm formation and function (Shrout and Nerenberg, 2012; see more details on this

system in Section 4.2.5.

4.2.3.8 Other Factors. A number of other factors (pH, redox potential, TOC,

water temperature, hardness) control the growth of microorganisms on pipe surfaces

(Olson andNagy, 1984). Temperature affects directly and indirectly the rate of biofilm

formation. A study of biofilm growth in pipes made of different materials showed that

biofilm accumulation was higher at 35◦C than at 15◦C (Silhan et al., 2006). Bacterial

growth generally increases when temperature is above 15◦C although psychtrophic

iron bacteria can grow below this temperature.

4.2.4 Biofilm Ecology

Biofilms allow microorganisms to persist and grow in hostile environments such as

WDSs under low nutrient conditions. In this protected environment, EPSs provide a

diffusional barrier against disinfectants and other deleterious chemicals. EPS increase

the resistance of biofilm and any detached biofilm clusters to chlorine (Xue et al.,

2013b). Using model WDSs, it was found that during the initial stages of biofilm

formation, the 16S RNA sequences in biofilms are similar to those found in the bulk

solution. However, DNA- and RNA-based fingerprints of 20-year-old biofilms show

a higher diversity in biofilm communities than in bulk water (Henne et al., 2012).

The higher diversity in biofilms than in bulk water was also observed in an exper-

imental WDS. Alphaproteobacteria dominated in the bulk water while beta- and

gammaproteobacteria (which include many primary and opportunistic pathogens)

were predominant in biofilms (Douterelo et al., 2013). Microcolonies are formed

following attachment of single cells to the pipe surface. Species diversity increases

thereafter, and biofilms may reach steady state and high ecological diversity, includ-

ing both heterotrophs and autotrophs, only after 2–3 years (Berry et al., 2006;Martiny

et al., 2003). Molecular techniques are now revealing the bacterial diversity in drink-

ing water and biofilms (Kahlisch et al., 2010; Lee et al., 2010; Revetta et al., 2010;

Schmeisser et al., 2003). Molecular probing showed the presence of gram-positive

bacteria as well as alpha-, beta-, and gamma-proteobacteria. Bacteria generally found

in drinking WDSs include Pseudomonas, Acinetobacter, Flavobacterium, Alcali-genes, Aeromonas,Moraxella, Enterobacter,Citrobacter, Sphingomonas,Klebsiella,Burkholderia, Xanthomonas, Methylobacterium, and Bacillus (Berry et al., 2006;

Block et al., 1997). Examination of biofilms from household water meters showed

that the major bacterial genera were Sphingomonas (alpha-proteobacteria), Acidovo-rax, Methylophilus (beta-proteobacteria), and Lysobacter (gamma-proteobacteria).Examination of the Ann Arbor drinking water by 16S rRNA gene sequencing also

showed that most of the bacteria were proteobacteria and belonged to Acidovorax,Variovorax, Sphingopyxis, Ralstonia, and Novosphingobium (Figure 4.5; Lee et al.,

2010). Another study in Ohio, using similar techniques, showed the presence of

proteobacteria, cyanobacteria, actinobacteria, bacteroidetes, and planctomycetes but

57.6% of the sequences were hard to classify (Revetta et al., 2010).

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100 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

Bacillus (3%)

E. Coli (3%)

Mesorhizobium

(3%)

Novosphigobium

(6%)

Ralstonia

(6%)

Sphingopyxis

(15%)

Variovorax (42%)

Acidovorax (21%)

Figure 4.5 Bacterial diversity in drinking water. Pie chart shows the relative diversity of

each genus identified by 16S rRNA gene sequencing. Source: Lee et al. (2010). Water Res. 44:

5050–5058.

Bacterial community composition changes following chlorine disinfection as

alpha-, beta-, and gamma-proteobacteria decreased after chlorination (Poitelon et al.,

2010). Community composition is also influenced by the type of disinfectant used

(Hong et al., 2010; Poitelon et al., 2010; Williams et al., 2004; Yu et al., 2010). It is

known that nutrient-depleted environments, such as marine waters and groundwater,

harbor ultramicrocells (UMCs) that pass through 0.2 μm filters (Velimirov, 2001).

Such ultramicrocells were detected in chlorinated drinking water and are part of the

biofilm community. Analysis of 16S rRNA sequences showed that UMC belong to

the Proteobacteria, Firmicules, and Actinobacteria. Some of the UMCs are potential

opportunistic pathogens, such as Stenotrophomonas maltophilia or Microbacteriumsp., that cause infections in immunocompromised patients in hospital setting (Silbaq,

2009).

4.2.5 Gene Exchange and Quorum Sensing in Biofilms

The proximity of microbial cells in biofilms offers opportunities for communication

and exchange of metabolic products and genetic material (e.g., antibiotic resistance

genes) between microorganisms (Cvitkovitch, 2004; Wuertz, 2002; Xi et al., 2009).

The use of modern techniques such as confocal laser scanning microscopy and

fluorescent-labeled (e.g., tagging with green fluorescent protein) plasmids has facili-

tated the study of gene transfer in biofilms and in the environment in general. These

methods have generally shown that gene transfer rates were much higher than those

determined by plating on selective media. In addition to gene transfer by conjugation

and transformation, biofilms can also be a suitable environment for gene transfer via

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BIOFILM DEVELOPMENT IN WDSs 101

transduction. An example is the transfer of the Shiga toxin (Stx) gene to E. coli byan Stx-encoding bacteriophage (Solheim et al., 2013).

Much work is presently being done on the molecular mechanisms of biofilm

development. For their survival and interactions in biofilms, bacteria are able to

communicate with cells of the same species or with other prokaryotic and eukaryotic

species. This cell-to-cell communication mechanism, also called “bacterial twitter,”

is quorum sensing which requires a threshold cell density for its triggering. It helps

microorganisms in biofilm development and function and improves their survival by

increasing their access to nutrients and defense against competing microorganisms

(Boyle et al., 2013; de Kievit, 2009; Shrout and Nerenberg, 2012; Williams et al.,

2007). Several quorum sensing bacteria has been found in water and wastewater

treatment systems. These include, among others,Aeromonas,Arcobacter, Legionella,Pseudomonas, Sphingomonas,Vibrio, andNitrobacter (Shrout andNerenberg, 2012).

Quorum sensing diffusible signaling molecules (e.g., N-acyl homoserine lactone),called autoinducers, play an important role in density-dependent gene expression and

biofilm differentiation. Bacteria undergo changes when the signaling molecule has

reached a threshold concentration and when the bacterial population has reached a

certain density. There are three major classes of signaling molecules: acyl homoser-

ine lactones (AHLs), peptides, and LuxS/autoinducer 2. Antibiotics can also func-

tion as signaling molecules at relatively low concentrations (Fajardo and Martınez,

2008). AHL signals are emitted by gram-negative bacteria and their activity was

demonstrated in single-species biofilms (e.g., Pseudomonas aeruginosa) and in nat-urally occurring biofilms (McLean et al., 1997). Peptides are the signaling molecules

in gram-positive bacteria. Autoinducer-2 (AI-2) is another cell-to-cell signaling

molecule that may function in cell communication in some bacteria (Winzer et al.,

2002). Some other functions regulated by the AI-2 signal are biofilm formation

in Bacillus cereus, motility in Helicobacter pylori, virulence factor production in

P. aeruginosa, or susceptibility to antibiotics in Streptococcus anginosus (Pereiraet al., 2013). In addition to biofilm formation, quorum sensing in both gram-negative

and gram-positive bacteria is also known to be involved in the regulation of several

activities, such as virulence, motility, EPS production, sporulation, production of sec-

ondary metabolites such as antibiotics, conjugation, symbiosis, and interspecies com-

petition and other survival strategies (Miller and Bassler, 2001; Withers et al., 2001).

4.2.6 Biofilm Detachment from Surfaces

Biofilm detachment from surfaces releases microorganisms, including pathogens,

which may colonize other surfaces. It affects the occurrence of waterborne diseases

resulting from the transport of opportunistic pathogens to the consumer’s faucet.

Detachment occurs following erosion (detachment of single cells), sloughing (detach-ment of large pieces of biofilm, sometimes extending all the way to the substratum),

scouring following abrasion or scraping and grazing by predators (Percival et al.,

2000; Rittmann, 2004; Rittmann and Laspidou, 2002).

Biological processes also involved in biofilm detachment, include, among other

factors, a decrease in EPS production, rhamnolipids, anti-biofilm poylysaccharides,

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102 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

and predation on biofilm microorganisms. Detachment events are genetically regu-

lated (Rendueles et al., 2013; Schooling et al., 2004; Stoodley et al., 2002).

The net accumulation of biofilms on surfaces is described by the following equation

(Rittmann and Laspidou, 2002).

Xf Lf =YJ

b + bdet(4.1)

where;

Xf = biomass density (mg biomass/cm3)

Lf = biofilm thickness (cm)

Y = true yield of biomass (mg biomass/mg substrate)

J = substrate flux (mg substrate/cm2-day)

b = specific decay rate of biofilm microorganisms (d−1)bdet = specific detachment rate (d−1)

Biofilm accumulation increases when the substrate is increased or when detach-

ment is decreased. Under steady state conditions, the average specific growth rate

(μave) is equal to the specific detachment rate (bdet).Slow growing microorganisms (e.g., methanogens) are protected from detachment

because they live in the deeper layers of the biofilm.

There are several factors affecting biofilm detachment (Pedersen, 1990; Rittmann

and Laspidou, 2002). Detachment is:

� increased by tangential flow shear stress which is related to the detachment rate

(at least for smooth surfaces);� increased by axial force on the biofilm, due to abrasion and to pressure changes

caused by turbulent changes and expressed by the Reynolds number Re;� decreased by surface roughness;� affected by physiological parameters such as EPS content (higher detachment

at low EPS) or biofilm density (lower detachment for dense biofilms).

4.2.7 Some Methods Used in Biofilm Study

Several physical, chemical, biochemical, molecular, andmicrobiological methods are

available for biofilms study. Some of these methods, including molecular techniques,

are summarized in Table 4.1 (Broschat et al., 2005; Characklis et al., 1982; Lazarova

and Manem, 1995; Neu and Lawrence, 2002; Percival et al., 2000; Schaule et al.,

2000; Schmidt et al., 2004).

In the cultivation-based method, a low nutrient medium, designated as R2A agar,

is recommended to determine the heterotrophic plate count (HPC) in environmental

samples such as drinking water or groundwater. However, this approach detects only

from 0.1% to 10% of the total bacterial counts in most aquatic environments (Pickup,

1991). Microbial biomass in biofilms can be measured via determination of specific

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BIOFILM DEVELOPMENT IN WDSs 103

TABLE 4.1 Some methods used for biofilm study

Type Analytical Method

Microscopy Use of light microcopy, fluorescence microscopy, scanning

confocal laser microscopy, scanning electron microscopy,

environmental scanning electron microscopy, transmission

electron microscopy, atomic force microscopy.

Image analysis Image structure analyzer, time lapse imaging

Direct

measurement of

biofilm quantity

Biofilm thickness (using optical methods, image analysis,

thermal resistance, quartz crystal microbalance)

Biofilm mass (total cell count via staining with Acridine Orange

or DAPI, crystal violet assay)

Indirect

measurement of

biofilm quantity

Extracellular polymeric substances (EPS)

Specific biofilm constituents (polysaccharides, proteins, nucleic

acids)

Total organic carbon (TOC)

Total proteins

Peptidoglycans

Lipid biomarkers

Microbial activity

within biofilms

Viable cell count (plate counts, preferably in low nutrient

medium such as R2A agar, Live/Dead BacLightTM)

Active bacteria: direct viable count (DVC)

DVC-FISH

ATP

Lipopolysaccharides

Substrate removal rate

Dehydrogenase activity (e.g., TTC, INT, or CTC dyes) or

esterases such as carboxyfluorescein diacetate (CFDA).

Oxygen uptake rate (OUR)

DNA

rRNA

mRNA

Microbial

observation and

identification

Immunofluorescence (monoclonal or polyclonal antibodies)

FISH (fluorescent in situ hybridization)mRNA amplified by polymerase chain reaction

Green fluorescent protein (GFP)

Use of commercial fluorochromes such as DAPI, acridine

orange, SYTOX Green, PicoGreen and propidium iodide, or

FUN-1 (for fungi)

Microenvironment Fluor conjugates for determining diffusion and permeability in

biofilms

Use of microelectrodes for determining pH, temperature, O2,

NH4, NO3, NO2, H2S, and CH4 within the biofilm

Other methods Mass spectroscopy

Infrared spectroscopy

X-ray spectroscopy

Source: Adapted from Broschat et al. (2005); Characklis et al. (1982); Lazarova and Manem (1995);

McLean et al. (2004); Mezule et al. (2013); Neu and Lawrence (2002); Percival et al. (2000); Schaule

et al. (2000); Schmidt et al. (2004).

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104 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

cell biochemical constituents such as ATP, DNA, RNA, proteins, lipid biomarkers,

bacterial cell wall components, or photosynthetic pigments (Sutton, 2002).

Total cell counts in biofilms are difficult due to the presence of aggregated cells.

This task is made easier by using modern techniques such as scanning confocal

microscopy (SCLM) which gives information about the biofilm structure. The non-

destructive SCLM in conjunction with 16S rRNA-targeted oligonucleotide probes

help to show the heterogeneous structure of biofilms and their complex biodiversity

by giving information on the distribution and activity of specific groups of microor-

ganisms in biofilms (Costerton et al., 1995; Schramm et al., 1996). rRNA-targeted,

fluorescently labeled oligonucleotide probes are useful in identifying biofilmmicroor-

ganisms and community composition (Kalmbach et al., 2000). Other nondestructive

microscopic techniques include epifluorescence microscopy using fluorogenic dyes

(e.g., acridine orange or 6-diamidino-2-phenylindol known as DAPI), transmission

electron microscopy (TEM), scanning electron microscopy (SEM), and environ-

mental SEM (ESEM) which allows the observation of the specimen in its own

hydrated state.

Microelectrode probes give information on the physicochemical properties (pH,

temperature, O2, NH4, NO3, NO2, H2S, CH4) of the biofilm microenvironment

(Burlage, 1997). These microelectrodes have helped shed light on the distribution

of nitrifiers and sulfate reducers along the depth of biofilms. Microelectrodes for

biofilm study will be replaced in the future with fiber-optic microsensors (Beyenal

et al., 2000).

Microbial activity within biofilms can also be assessed by measuring enzyme

activity (e.g., esterase or dehydrogenase activity), reduction of tetrazolium salts such

as cyanoditolyl tetrazolium chloride (CTC), 2-p-(iodophenyl)-3-(p-nitrophenyl)-5-tetrazolium chloride (INT), triphenyl tetrazolium chloride (TTC), or resorufin by

biofilmmicroorganisms. Fluorescein diacetate (FDA) or carboxyfluorescein diacetate

(CFDA) are used to measure esterase activity and the active fluorescent cells are

counted under a fluorescent microscope. Cell viability can also be determined by the

LIVE/DEAD BacLight kit, or special dyes which measure membrane integrity (e.g.,

SYBR Green propidium iodide). The fluorescence intensity can be determined via

flow cytometry (Berney et al., 2007, 2008; Cerca et al., 2011).

Several techniques have been proposed for gaining information about biofilm

formation and thickness. Some of the methods are (Broschat et al., 2005; Milferstedt

et al., 2006; Nivens et al., 1995; Reipa et al., 2006):

� Quartz crystal microbalance (QCM). This nondestructive technique measures

biomass changes on a quartz crystal. Both the quartz oscillator frequency (f)and resistance (R) are monitored during biofilm formation and the data are

expressed as ΔR/Δf which reflects changes in the viscoelastic properties of thebiofilms. QCM was used in the long-term monitoring of biofilm formation by

Pseudomonas cepacia and P. aeruginosa.� Optical methods: Early on, biofilm thickness was estimated by focusing a light

microscope on the top and bottom layers of a biofilm. A more recent approach

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GROWH OF PATHOGENS AND OTHER MICROORGANISMS IN WDSs 105

consists of exposing the biofilm to a desktop scanner and then following with

image analysis. The gray level of the images is well correlated with the biofilm

biomass. A reflectance assay was considered to determine biofilm formation of

14 Enterococcus isolates on opaque and nonopaque surfaces� Crystal violet assay: This method consists of staining dry biofilms with 0.1%crystal violet, washing with buffer to remove excess dye, releasing the dye from

the biofilm with 95% ethanol and measuring the absorbance at 595 nm.

4.3 GROWH OF PATHOGENS AND OTHER

MICROORGANISMS IN WDSs

Pathogen accumulation in biofilms of WDSs is an important public health issue as

it may contribute to the spread of waterborne diseases. Pathogens enter the WDS

through insufficient water treatment or through leaks. They grow in WDSs and

colonize pipe inner walls, connections, tubercles, and dead ends. However, biofilm

growth over surfaces is not continuous as noncolonized spots are observed. Bacterial

accumulation preferably occurs in the colonized area and increases with biofilm age

(Paris et al., 2009).

4.3.1 Earlier Studies on the Microbiology of Distribution Systems

The presence of a wide range of microorganisms (eubacteria, filamentous bacteria,

actinomycetes, diatoms) was demonstrated in distribution pipes by SEM. Some of

the bacteria were seen attached to the surfaces by means of extracellular fribrillar

materials (Ridgway andOlson, 1981; Ridgway et al., 1981). Turbercles provide a high

surface area for microbial growth and protect microorganisms from the lethal action

of disinfectants. Turberculated cast iron pipe sections from the WDS in Columbus,

Ohio, metropolitan area harbored high numbers of aerobic and anaerobic bacteria

(e.g., sulfate reducing bacteria). Some samples contained up to 3.1 × 107 bacteria per

gram of tubercle material (Tuovinen and Hsu, 1982). Several investigators have also

reported the proliferation of iron and manganese bacteria that grow attached to pipe

surfaces and, subsequently, cause a deterioration of water quality (e.g., pipe clogging

and color problems). Attached iron bacteria such as Gallionella have stalks that arepartially coveredwith iron hydroxide. This was confirmed byX-ray energy-dispersive

microanalysis (Ridgway et al., 1981).

4.3.2 Opportunistic Bacterial Pathogens

As mentioned in Chapter 1, this group includes genera such as Pseudomonas,Aeromonas, Klebsiella, Flavobacterium, Enterobacter, Citrobacter, Serratia, Acine-tobacter, Proteus, Providencia, L. pneumophila, S. maltophilia, and nontubercular

mycobacteria (NTM) (Sobsey and Olson, 1983; van der Wielen and van der Kooij,

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106 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

2013). They are of health concern to the young, the elderly, and immunocompromised

consumers. The routine occurrence of these opportunistic pathogens in distribution

systems have been documented in the literature (Keevil, 2002). For example, most

Aeromonas isolates from 13 Swedish drinking WDSs produced virulence factors

such as cytotoxins, suggesting potential public health problems (Kuhn et al., 1997).

Sphingomonas spp. can also be found in water distribution biofilms. They are oftenoverlooked due to their slower growth than other members of the HPC (Koskinen

et al., 2000). They have been isolated from activated carbon and sand filters, and are

potential reservoirs of antibiotic-resistant bacteria in drinking water (Vaz-Moreira

et al., 2011). Biofilms can also be a potential reservoir for H. pylori which can sur-vive in the viable but nonculturable (VBNC) state (Linke et al., 2010; Percival and

Thomas, 2009). The VBNC state is a bacterial response to stress (e.g., nutritional

stress, exposure to disinfectants and other toxicants such as heavy metals). A wide

range of bacteria are now known to enter in the VBNC state when exposed to envi-

ronmental stress (Oliver, 2005). Biofilmsmay also harbor antibiotic-resistant bacteria

(Schwartz et al., 2003; see also Chapter 1).

4.3.3 Nontubercular Mycobacteria (NTM)

The genusMycobacterium includes pathogenic species such asM. tuberculosum and

M. leprae, and NTM which are found in soil and aquatic environments and are con-

sidered as opportunistic pathogens (Falkinham, 2002; Falkinham et al., 2001). NTM

were detected around the world in 21% to more than 80% of samples from WDSs

(see review by Vaerewijck et al., 2005). A more recent survey in two chloraminated

WDSs in Florida and Virginia showed that the percent occurrence ofMycobacteriumspp. was 93.7% and 94.4%, respectively. The percent occurrence of M. avium was

10% and 8.9%, respectively (Wang et al., 2012b). In Germany and France, mycobac-

terial species were found in 90% of biofilm samples taken from water treatment

plants (Schulze-Robbecke et al., 1992). A survey of seven water treatment plants in

the Netherlands showed that NTM were detected in all distributed drinking water

samples and their levels were much higher in distributed water than in treated water

(van der Wielen and van der Kooij, 2013). Approximately one-third of the 100

known mycobacterial species have been detected in WDSs. In the Netherlands, sev-

eral mycobacterial species (M. peregrinum, M. salmoniphilum, M. llatzerense, andM. septicum) were identified in distributed water and in shower water (van Ingen

et al., 2010). The primary Mycobacterium species detected in biofilms of WDSs

in China (Guangzhou and Beijing) were M. arupense and M. gordonae (Liu et al.,2012a). Mycobacterium avium subsp. paratuberculosis (MAP), a possible causative

agent of Crohn’s autoimmune disease in humans, was detected, via PCR, in finished

drinking water (Aboagye and Rowe, 2011; Beumer et al., 2010). This opportunistic

pathogen persists for several weeks in biofilms and its survival is much higher than

that of E. coli (Lehtola et al., 2007; Torvinen et al., 2007). The growth of M. aviumin WDSs depends on the level of available organic carbon (Falkinham et al., 2001;

LeChevallier, 2004).

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GROWH OF PATHOGENS AND OTHER MICROORGANISMS IN WDSs 107

4.3.4 Legionella in Hot-Water Tanks and Distribution Systems

Due to airborne transmission through showering, much work has been carried out on

the survival and growth of Legionellae in potable WDSs and plumbing in hospitals

and homes (Colbourne et al., 1988; Felfoldi et al., 2010; Muraca et al., 1988). A

recent survey showed that the percent occurrence of Legionella spp. was 67.5% in

a Florida WDS and 30% in a Virginia system (Wang et al., 2012b). Furthermore,

legionellae appear to be more resistant to chlorine than E. coli (States et al., 1989),and small numbers may survive in distribution systems that have been judged to be

microbiologically safe. Under high shear turbulent-flow conditions, survival of L.pneumophila and Mycobacterium avium is much higher than that of E. coli whichis traditionally used as an indicator (Lehtola et al., 2007). Legionellae may also

grow to detectable levels inside hot-water tanks in hospitals and homes and thus

pause a health threat (Stout et al., 1985; Witherell et al., 1988). The Legionellaisolates responsible for nosocomial Legionnaires’ disease corresponded to isolates

from potable water (Garcia-Nunez et al., 2008). Legionella survives well at 50◦C

and may even grow and multiply in tap water at 32–42◦C (Dennis et al., 1984;

Yee and Wadowsky, 1982). The enhanced survival and growth in these systems

have also been linked to stagnation (Ciesielski et al., 1984), stimulation by rubber

fittings in the plumbing system (Colbourne et al., 1988), and trace concentrations

of metals such as Fe, Zn, and K (States et al., 1985, 1989). It was found that

sediments found in WDSs and tanks (i.e., scale and organic particulates) and the

natural microflora significantly improved the survival of Legionella pneumophila.Sediments indirectly stimulate Legionella growth by promoting the growth of com-mensalistic microorganisms (Stout et al., 1985; Wadowsky and Yee, 1985). For

example, Mycobacterium chelonae, an opportunistic bacterial pathogen, can have apositive effect on the cultivability of L. pneumophila and H. pylori in biofilms (Giaoet al., 2011).

Protozoa, mostly amoebas and ciliates, play a role in the survival of Legionellain water distribution pipes. Biofilms and the presence of protozoa (e.g., Acan-thamoeba spp.) play an essential role in the survival, proliferation, and pathogenicityofLegionella in drinkingwater (Corsaro et al., 2010; Lau andAshbolt, 2009). Biofilmsgrowing in in-premise plumbing harbor Legionella cells as well as protozoan hostswhich help in the propagation of this pathogen. The biofilm-associated Legionella isdetached and aerosolized during showering and can be subsequently inhaled.

Several methods have been considered for controlling Legionella in water: super-chlorination (chlorine concentration of 2–6 mg/L), chloramination, maintaining the

hot-water tanks at temperatures above shock treatment at 70◦C for 30minutes, ultravi-

olet irradiation (Antopol and Ellner, 1979; Knudson, 1985; Kool et al., 2000; Muraca

et al., 1987; Stout et al., 1986), biocides (Fliermans and Harvey, 1984; Grace et al.,

1981; Soracco and Pope, 1983), and alkaline treatment (States et al., 1987). There

are less Legionella nosocomial infection outbreaks in hospitals that use monochlo-ramine than those using free chlorine (Flannery et al., 2006; Heffelfinger et al., 2003;

Kool et al., 1999). Legionella can, however, become heat-resistant after repeated heattreatments (Allegra et al., 2011). The control of legionellae in potable water systems

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108 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

may also be achieved via the control of protozoa which may harbor these pathogens

(States et al., 1990).

4.3.5 Antibiotic-Resistant Bacteria and Antibiotic Resistance Genes

Antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARG) to sev-

eral antibiotics (amoxilline, chloramphenicol, ciprofloxacin, gentamicin, rifampin,

sulfisoxasole, tetracycline) were found in several finished and tap water samples.

The ARB levels were often higher in tap water than in finished water, indicating

that WDSs can serve as a reservoir for ARB and ARG (Xi et al., 2009). Antibiotic

resistance genes have been detected in biofilms from drinkingWDSs. Enterobacterial

ampC β-lactam-resistance and enterococcal vanA vancomycin-resistance genes were

detected in drinking water biofilms although no enterobacteriaceae or enterococci

were detected in those samples. However, the Staphylococcal mecA methicillin-

resistance gene was not found in drinking water biofilms (Schwartz et al., 2003).

Several studies have shown that chlorination of water and wastewater induces

selection for ARB (Murray et al., 1984; Shrivastava et al., 2004). The mechanism of

this selection is not well known.

However, at the present time, we do not know if increased resistance to antibi-

otic present a significant risk to consumers, especially the young, the elderly, and

immunocompromised people.

Table 4.2 lists some human pathogens and opportunistic pathogens detected in

biofilms of domestic plumbing systems (Eboigbodin et al., 2008).

4.3.6 Protozoan Parasites

Biofilms can also serve as reservoirs for protozoan parasites cysts and oocysts. The

trapping and concentration of C. parvum oocysts and their survival in biofilms was

demonstrated under laboratory conditions (Rodgers and Keevil, 1995). Free-living

TABLE 4.2 Some human pathogens detected in biofilms from domestic plumbingsystems

Pathogen Comments

Legionella pneumophila Causes Legionnaires’ disease

Legionella bozemanii Causes human pneumonia

Helicobacter pylori Causes gastric diseases including cancer

Pseudomonas sp. Some species (e.g., P. aeruginosa, P. maltophilia) arehuman pathogens

Sphingomonas sp. Some species cause nosocomial infections in humans

Micrococcus kristinae Found in catheter-associated bacteremia

Corynebacterium sp. Part of human skin flora. Some species are pathogenic

Acanthamoeba keratitis Can cause infection in the cornea

Source: Adapted from Eboigbodin et al. (2008). Amer. Water Works Assoc. J. 100 (10): 131–137.

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SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS IN DRINKING WATER 109

pathogenic protozoa, such as Naegleria fowleri, may also find refuge in biofilms,

particularly in stagnant areas of the distribution network. There is experimental

evidence thatN. fowleri can colonize and persist for months in pipe biofilms, althoughnone was detected in two full-scale distribution networks (Biyela et al., 2012).

The cysts and oocysts can subsequently be resuspended in WDSs by biofilm

sloughing following an increase in water flow (Helmi et al., 2008; Puzon et al., 2009;

Searcy et al., 2006).

4.3.7 Enteric Viruses

Enteric viruses are occasionally detected in treated drinking water (Bitton et al.,

1986). Laboratory studies have shown that, following their entry into the WDS,

viruses may adsorb to biofilms where they may survive for relatively long peri-

ods. A similar phenomenon was observed in wastewater systems where viruses

(noroviruses, enteroviruses, FRNA phages) were found to attach and persist in

biofilms for longer periods than in wastewater (Skraber et al., 2009). However, only

one of three field studies of virus monitoring in biofilms showed the presence of infec-

tious enteroviruses (Vanden Bossche, 1994). Similarly to other pathogens, pathogenic

viruses may contaminate drinking water through sloughing of biofilm pieces.

4.4 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

IN DRINKING WATER TREATMENT AND DISTRIBUTION

The development of biofilms on surfaces can be beneficial or detrimental to processes

in water and wastewater treatment plants. Trickling filters and rotating biological

contactors are examples of processes that rely on microbial activity in biofilms to

treat wastewater.

4.4.1 Advantages

Some advantages offered by biofilms are (Rittmann, 1995b; Shrout and Nerenberg,

2012)

� The fact that anytime a solid surface is exposed to water is followed by biofilm

formation demonstrates well the benefits of biofilms to microorganisms.� Biofilms act as biocatalysts in natural systems for the self-purification of surface

waters and groundwater, and in engineered fixed-film processes (e.g., trickling

filters, rotating biological contactors, biofilters in water purification, riverbank

filtration, membrane-based biofilm reactors) for the treatment of water and

wastewater.� They are desirable whenever microorganisms must be retained (i.e., need for a

high mean cell residence time) in a system with a short hydraulic retention time,

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110 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

as is the case for biological treatment of drinking water which involves the pro-

cessing of large volumes of water with very low concentrations of biodegradable

compounds.� Many of the processes used in drinking water treatment allow biofilm formation

and include, among others, granular activated carbon, slow and rapid sand filters,

and biological water treatment.

4.4.2 Disadvantages

However, biofilms can cause problems in water treatment and distribution systems

(Rittmann, 1995b, 2004; Smith, 2002; van der Wende and Characklis, 1990):

� Because of mass-transport resistance, bacteria are exposed to lower concentra-

tions of substrates than in the bulk liquid.� Biofilm accumulation increases fluid frictional resistance in distribution

pipelines (Figure 4.6; Bryers and Characklis, 1981), leading to an increase

in pressure drop and to reduced water flow if the pressure drop is held con-

stant. The microbial community structure of the biofilm also affects frictional

resistance. A predominantly filamentous biofilm appears to increase frictional

resistance (Trulear and Characklis, 1982).� Biofouling decreases flow rates in membrane-based water treatment processes.

The biofouling is due to the overproduction of EPSs mainly composed of

polysaccharides.

GROWTHINDUCTION

TIME

PLATEAU

Biofilm mass

or thickness

Frictional

resistance

Heat transter

resistance

Figure 4.6 Increase in heat transfer resistance and frictional resistance resulting from biofilm

growth. Source: Bryers and Characklis (1981). Water Res. 15: 483–491.

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SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS IN DRINKING WATER 111

� Anaerobic conditions lead to the production of H2S, a toxic gas characterized

by a rotten-egg odor.� Biofilms have an impact on public health and are the cause of persistent infec-

tions (Costerton, 1999). They are conducive to the accumulation of pathogens

and parasites in distribution pipes biofilms which are habitats for opportunistic

pathogens such as Legionella andMycobacterium and lead to infections related

to implants and dental plaques. Biofilms are involved in 80% of chronic inflam-

matory and infectious diseases due to pathogenic bacteria (Sauer et al., 2007).

Biofilms developing on implanted medical devices harbors microbial pathogens

such as Candida albicans, Staphylococcus aureus, P. aeruginosa, Kelbsiellapneumoniae, and Enterococcus spp., and E. coli. Most of the circulatory and

urinary tract infections are due to biofouled implanted devices. These infec-

tions increase patient morbidity and mortality and the cost of medical care.

Biofilms associated with medical implants are often resistant to antibacterial

and antifungal drugs (Anwar and Costerton, 1992; Donlan, 2002; Thomas et al.,

2004).� Corrosion problems: Biocorrosion of pipes is associated with biofilm growth.

Biofilm accumulation is associated with corrosion of iron pipes where Fe2∗

serves as an electron donor. Complaints about red and black waters, due to the

activity of iron- and manganese-oxidizing bacteria (e.g., Gallionella, Hyphomi-crobium) (Rittmann, 2004).

� Resistance of biofilm microorganisms to biocides: Biofilm microorganisms can

be up to 1500 times more resistant to biocides and antibiotics than freely sus-

pended bacteria (Sauer et al., 2007). Increase in resistance to chlorine dis-

infection contributes to the regrowth of indicator and pathogenic bacteria in

distribution systems. It was suggested that this increased resistance may be due

to diffusional resistance of the biofilmmatrix as measured with a chlorinemicro-

electrode (de Beer et al., 1994), the protective effect by extracellular polymeric

materials, the presence of efflux pumps that export biocides, antibiotics, and

other toxic substances to the bacterial surrounding medium (Marquez, 2005),

selection of biofilmmicroorganismswith enhanced resistance to the disinfectant,

attachment of bacteria to biological (e.g., macroinvertebrates and algal surfaces)

and nonbiological surfaces (particles, activated carbon) or cell surface hydropho-

bicity (Steed and Falkinham, 2006). Furthermore, multispecies biofilms appear

to be more resistant to disinfection than single-species ones (Simoes et al.,

2010). As shown for activated carbon, biofilm bacteria appear to be more resis-

tant to residual chlorine and monochloramine than suspended bacteria (Berry

et al., 2009; LeChevallier et al., 1988). The protective effect of biofilms toward

pathogens exposed to chlorine was also demonstrated forMycobacterium aviumand Mycobacterium intracellulare (Steed and Falkinham, 2006). Particulates,

including particulate organic matter, protect pathogenic microorganisms from

disinfectant action (Hoff, 1978). It was found that Enterobacter cloacae, in thepresence of particulates fromWDSs, is protected from chlorine action as a result

of its attachment to the particles (Herson et al., 1987). Chloramines appear to

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112 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

be more efficient in biofilm control than free chlorine (HOCl or OCl−) (seeChapter 3). This may be due to the lower affinity of chloramines for bacte-

rial polysaccharides (LeChevallier et al., 1990; van der Wende and Characklis,

1990). It was proposed that, for biofilm control, free chlorine should be used

as a primary disinfectant but the residual should be converted to chloramine

(LeChevallier et al., 1990). The increased resistance of biofilm microorganisms

to chlorine applies to other antibacterial agents such as antibiotics. The free

chlorine residual decreases as the water flows through the distribution system.

In a laboratory study, silver (100 μg/L) used as a biocide did not have any signif-icant effect on preventing biofilm formation on PVC and stainless steel surfaces

(Sylvestry-Rodriguez et al., 2008).

4.5 BIOFILM CONTROL AND PREVENTION

Biofilms can be controlled or removed by mechanical, chemical, or enzymatic means

(Brisou, 1995). The drinking water industry has a few options for controlling biofilm

growth in WDSs (Camper et al., 2003; Rittmann, 2004):

� Mechanical cleaning: It is combinedwith the use of biocideswhen the biofouling

is severe.� Maintain a proper level (1 mg/L as Cl2) of chlorine residual throughout the

WDS to reduce biofilm accumulation. The chlorine level cannot exceed 4 mg/L

as Cl2. Maintaining a chlorine residual is, however, difficult to achieve in large

systems and cannot prevent biofilm growth in the presence of high levels of

electron donors.� Obtain a biostable water by removing or drastically reducing the level of organic

and inorganic electron donors in treated water. This can be accomplished by

using membrane filtration, enhanced coagulation, or biological filtration (see

Chapter 6).� Consider the effect of the pipe material: Replace deteriorating iron pipes with

other materials (e.g., PVC or CPVC pipes). However, one should be concerned

about the leaching and accumulation of the monomer, vinyl chloride, a proven

human carcinogen (Walter et al., 2011).� Potential “jamming” of quorum sensing in biofilms (Barraud et al., 2009;

Hentzer et al., 2004; Shrout and Nerenberg, 2012): Attempts to interrupt quorum

sensing in biofilms are potentially a useful strategy in the control of infectious

diseases. As a result of the emergence of antibiotic resistance in bacteria, there

is presently a quest for quorum sensing inhibitors to attenuate and control bacte-

rial infections (Hentzer et al., 2003). Laboratory studies have shown that several

chemicals can control biofilms by interrupting quorum sensing. Some examples

are 2,4-dinitrophenol, a furanone produced by red algae, nitric oxide to dis-

perse single- or multi-species biofilms, or protoanemonin, a natural compound

which inhibits quorum sensing in P. aeruginosa. The application of this research

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BIOFILM CONTROL AND PREVENTION 113

0

10

20

30

40

50

60

70

80

90

100

PAβNNMPT

Bio

film

fo

rma

tio

n r

ela

tive

to

co

ntr

ol w

ith

ou

t E

PIs

Figure 4.7 Effect of efflux pump inhibitors (T, NMP and PAβN) on biofilm formation in

Staphylococcus aureus. T = Thioridazine; NMP = 1-naphthylmethyl piperazine; PaβN =Phe-Arg-β Naphthylamide. Adapted from Kvist et al. (2008). Appl. Environ. Microbiol. 74:

7376–7382.

to WDSs under field conditions awaits further investigation. Quorum sensing

interruption by chemicals could be used in combination with other control mea-

sures to improve biofilm control. Somemicroorganisms make endogenous nitric

oxide (NO)which is generated byNO-synthase under the control of the nos gene.The presence of NO helped decrease the ability of P. putida to form biofilms

(Bobadilla Fazzini et al., 2013; Liu et al., 2012b; Njoroge and Sperandio, 2009).

There are also quorum enzymes or bacteria that release enzymes that inhibit

signaling in biofilms, leading to control of biofouling in membrane bioreactors

(Jiang et al., 2013; Kim et al., 2013b). As an example, a recombinant E. colithat produces N-acyl homoserine lactonase, inhibited biofouling in a membranebioreactor (Oh et al., 2012; Yeon et al., 2009a, 2009b). The feasibility of this

approach for controlling biofilms is yet to be demonstrated.� As regard the formation of biofilms on medical devices, great efforts are

being made to prevent their formation and to develop drugs to treat existing

biofilms. One original control method is to block quorum-sensing pathways in

order to prevent the formation of biofilms on medical devices. Bacteria also

have several chromosome-encoded efflux pumps that help export antibiotics,

antiseptics, heavy metals, solvents, detergents, and other biocides to the sur-

rounding medium (Martinez et al., 2009). These efflux pumps would lead to

increased resistance of biofilms to toxic chemicals, including disinfectants. The

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114 SOME ADVANTAGES AND DISADVANTAGES OF BIOFILMS

addition of efflux pumps inhibitors (e.g., thioridazine, phe-arg β-naphthylamide,1-naphthylmethyl piperazine) helped reduce biofilm formation by Staphylococ-cus aureus (Figure 4.7; Kvist et al., 2008). A wide range of chemicals are being

screened by biotechnological companies to find a way to control biofilms. Some

of these chemicals are the halogenated furanones produced by marine algae

(Schachter, 2003).� Antibiofilm polysaccharides: Some bacteria are able to produce antibiofilm

polysaccharides which can inhibit biofilm formation or destabilize preformed

biofilms. They potentially have useful applications in industrial and medical

fields and can help reduce the incidence of infections caused by biofilms on

medical devices (Rendueles et al., 2013).� Other biofilm control approaches are surface modification or application of an

electric current (Chiang et al., 2009; Wellman et al., 1996).

WEB RESOURCES

http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/

(biofilm online manual, Amer. Soc. Microbiology)

http://www.personal.psu.edu/faculty/j/e/jel5/biofilms/primer.html

(biofilm primer)

http://www.erc.montana.edu/

(Center for Biofilm Engineering, Montana State University, Bozeman)

http://www.nesc.wvu.edu/ndwc/articles/OT/SP01/History_Distribution.html

(history of drinking water distribution)

http://www.nottingham.ac.uk/quorum/

(general information about quorum sensing)

http://www.mrc-lmb.cam.ac.uk/genomes/awuster/wecb/

(gives many references on quorum sensing)

http://www.epa.gov/safewater/dwh/index.html

(Drinking water: EPA consumer information)

http://www.epa.gov/safewater/sdwa/index.html

(SDWA: Safe Drinking Water Act)

http://www.epa.gov/safewater/standards.html

(General information on drinking water)

http://www.cdc.gov/safewater/

(Safe Water System, Center for Disease Control and Prevention)

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